The present invention presents bispecific antibodies, the hybrid hybridomas which produce them, the parent hybridomas, the production and selection of the hybridomas and hybrid hybridomas and the purification of the bispecific antibodies. The specific examples are bispecific monoclonal antibodies which recognize both the human multiple-drug resistance (MDR) antigen, P-glycoprotein, and human Fc.gamma. receptor III (hFc.gamma.RIII). Background information as to each of the elements is presented below.
1. Hybridomas and Monoclonal Antibodies (MAB)
Antibodies are normally synthesized by lymphoid cells derived from B lymphocytes of bone marrow. Lymphocytes derived from the same clone produce immunoglobulin of a single amino acid sequence. Lymphocytes cannot be directly cultured over long periods of time to produce substantial amounts of their specific antibody. However, Kohler et al., 1975, Nature, 256:495, demonstrated that a process of somatic cell fusion, specifically between a lymphocyte and a myeloma cell, could yield hybrid cells which grow in culture and produce a specific antibody called a "monoclonal antibody" (hereinafter also referred to as "MAB"). The resulting hybrid cell was called a "hybridoma". A monoclonal antibody belongs to a group of antibodies whose population is substantially homogeneous, i.e. the individual molecules of the antibody population are identical except for naturally occurring mutations. Myeloma cells are lymphocyte tumor cells which, depending upon the cell strain, frequently produce an antibody themselves, although "non-producing" strains are known.
2. Multiple Drug Resistance In Cells
Certain cells are capable of developing resistance to drugs. Hamster, mouse and human tumor cell lines displaying multiple-drug resistance (MDR) have been reported. A major problem in the chemotherapy of cancer is the development of cross-resistance of some human tumors to multiple chemotherapeutic drugs. This type of multiple-drug resistance is accompanied by a decrease in drug accumulation and an increase in the expression of a multiple drug resistance protein, which is also known as P-glycoprotein or gp170. (Throughout this patent application, the term "P-glycoprotein" shall denote both P-glycoprotein and gp170). P-glycoprotein is a high molecular weight membrane protein (Mw 170-180 kDa) encoded by the MDR1 gene which is often amplified in MDR cells. The complete nucleotide sequence of the coding region of the human MDR1 gene and the complete corresponding amino acid sequence are disclosed in Patent Cooperation Treaty patent application, publication number WO 87/05943, priority date Mar. 28, and Aug. 1, 1986, "Compositions and methods for clones containing DNA sequences associated with multi-drug resistance in human cells," to Roninson, I. B. A method of isolating cDNA specific for P-glycoprotein is described in European Patent Application, Publication No. 174,810, date of publication, Mar. 3, 1986, "Multi-drug resistance in mammalian cell lines and isolation of determinant glycoprotein DNA", to Riordan, J. R.
While the "classical" MDR is based on P-glycoprotein, the "non-classical" MDR is based on other mechanisms, some of them as yet undefined. Throughout this patent application, the collective term "MDR phenotype" shall include both the classical -and non-classical MDR phenotypes. "MDR markers" or "MDR antigens" include P-glycoprotein and other antigens expressed solely or differentially on cells expressing the MDR phenotype. Different mutant cell lines exhibit different degrees of drug resistance. Examples of cell lines exhibiting the MDR phenotype have been selected for resistance to a single cytotoxic agent These cell lines also display a broad, unpredictable cross-resistance to a wide variety of unrelated cytotoxic drugs having different chemical structures and targets of action, many of which are used in cancer treatment. This resistance impedes the efficacy of drugs used in chemotherapy to slow down or decrease the multiplication of cancerous cells.
A monoclonal antibody that is capable of recognizing the K562/ADM adriamycin-resistant strain of a human myelogenous leukemia cell line K562 has been disclosed in European Patent Application, Publication No. 214,640 A3, "Monoclonal antibody in relation to drug-resistant cancers and productions thereof," to Tsuruo, T., published Mar. 18, 1987. This monoclonal antibody is produced by a hybridoma formed as a fusion product between a mouse myeloma cell and a spleen cell from a mouse that has been immunized with the K562/ADM strain.
3. Fc Receptors (FcRs)
Fc receptors are found on many cells which participate in immune responses. Fc receptors (FcRs) are cell surface receptors for the Fc portion of immunoglobulin molecules (Igs). Among the human FcRs that have been identified so far are those which recognize IgG (designated Fc.sub..gamma. R), IgE (Fc.sub..epsilon. R.sub.1), IgA (Fc.sub..alpha. R), and polymerized IgM/A (Fc.sub..beta.,.alpha. R). The different kinds of FcRs are found in the following cell types: Fc.sub..epsilon. R.sub.I (mast cells), Fc.sub..epsilon. R.sub.II (many leukocytes), Fc.sub..alpha. R (neutrophils), and Fc.sub..mu.,.alpha. R (glandular epithelium, hepatocytes), according to Hogg, N., 1988, Immun. Today, 9:185-86, "The structure and function of Fc receptors". The widely studied Fc.sub..gamma. Rs are central in cellular immune defenses, and are responsible for stimulating the release of mediators of inflammation and hydrolytic enzymes involved in the pathogenesis of autoimmune disease (Unkeless, J. C., 1988, Ann. Rev. Imm., 6:25 1-87, "Structure and function of human and murine receptors for IgG"). The Fc.sub..gamma. Rs provide a crucial link between effector cells and the lymphocytes that secrete Ig, since the macrophage/monocyte, polymorphonuclear leukocyte, and natural killer (NK) cell Fc.sub..gamma. Rs confer an element of specific recognition mediated by IgG. Id. Human leukocytes have at least three different receptors for IgG: hFc.sub..gamma. RI (found on monocytes/macrophages), hFc.sub..gamma. RII (on monocytes, neutrophils, eosinophils, platelets, possibly B cells, and the K562 cell line), and hFc.sub..gamma. RIII (on NK cells, neutrophils, eosinophils, and macrophages). Id.
4. Hybridoma 3G8
Hybridoma 3G8 is a murine hybridoma which secretes a mouse IgG1 MAB that recognizes human Fc.sub..gamma. RIII on human and chimpanzee leukocytes. For example, MAB 3G8 recognizes Fc.sub..gamma. RIII on neutrophils, monocytes, macrophages, and NK cells. MAB and hybridoma 3G8 are described in Unkeless, et al., supra, and was initially disclosed in Unkeless, J. C., et al., 1979, J. Exp. Med., 150:580-596.
5. Triomas, Hybrd Hybridomas, and Bispecific Antibodies
A bispecific antibody has binding sites for two different antigens within a single antibody molecule. Antigen binding may be simultaneous or sequential. Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies.
Triomas are generally formed from the fusion of a hybridoma and a lymphocyte. Hybrid hybridomas are generally formed by somatic cell fusion of two hybridomas. The hybridomas and lymphocytes each produce a mono-specific antibody, i.e., an antibody in which all binding sites recognize the same antigen. However, triomas and hybrid hybridomas may synthesize light and heavy chains of both parental types, and a bispecific antibody is composed of both light and heavy chains of both fusing partners. For an IgG producing cell, if the light and heavy chains of both kinds are made in equivalent amounts and combined randomly, at least one-eighth of the antibodies produced will be bispecific antibodies. However, in practice, due to preferential pairing of homologous light and heavy chains, many IgG/IgG fusions produce more than one-eighth bispecific antibody. It is also possible that a hybrid hybridoma or trioma may make little or no bispecific antibody, if heterologous heavy chain/heavy chain pairing is disfavored.
Examples of bispecific antibodies produced by a hybrid hybridoma or a trioma are disclosed in "Recombinant Monoclonal Antibodies", U.S. Pat. No. 4,474,893, to Reading, C. L., issued Oct. 2, 1984. Bispecific antibodies have been constructed which have both an anti-T cell and an anti-tumor antigen activity. Initially, a bispecific antibody conjugate was constructed by chemical means (Staerz et al., 1985, Nature, 314:628, and Perez, et al., 1985, Nature, 316:354). Later, hybridoma technology was employed (Staerz & Bevan, 1986, PNAS (USA), 83:1453, and Staerz & Bevan, 1986, Immunology Today, 7:241). The bispecific antibodies exert their effect by binding to both a tumor cell or other form of target cells, such as a virally infected cell, and to a T-cell thereby causing the destruction of the former by the latter.
Unlike the bispecific antibody prepared by the hybrid hybridomas technology described above, the chemically constructed bispecific antibody are prepared by chemically cross-linking heterologous Fab or F(ab').sub.2 fragments by means of chemicals such as heterobifunctional reagent succinimidyl-3-(2-pyridyldithiol)-propionate (SPDP, Pierce Chemicals, Rockford, Ill.). The Fab and F(ab').sub.2 fragments are obtained from intact antibody by digesting it with papain or pepsin, respectively. (For the procedures for chemically constructing such bispecific antibodies, see e.g., Karpovsky, B. et al., 1984, J. Exp. Med., 160:1686; Titus, J. A. et al., 1987, J. Immunol., 138:4018.) A chemically constructed bispecific antibody consisting of MAB 3G8 chemically cross-linked to a melanoma specific MAB could direct Fc.gamma.RIII bearing lymphocytes to kill melanoma cells both in vitro and in nude mice. Titus, J. A., et al., 1987, J. Immunol., 139:3153. Further, another chemically constructed bispecific antibody anti-CD3/MRK16, was reactive with P-glycoprotein on MDR cells and CD3 antigen on T-lymphocytes. The anti-CD3/MRK16 bispecific antibody was found to induce lysis of MDR tumor cells in vitro. Van Dijk, J. et al., Int. J. Cancer, 44:738 (1989). However, none of the above references discloses a bispecific antibody, produced by the hybrid hybridoma technology, which recognizes tumor cells exhibiting the MDR phenotype and cytotoxic cells with human Fc.gamma.RIIIs.